PROJECT SUMMARY/ABSTRACT Autophagy is a cellular recycling process with critical roles in aging and age-related diseases. Autophagy was originally described as a process mainly regulated at the post-translational level, but a role for transcriptional regulation has recently started to emerge. The relatively new concept of transcriptional regulation of the autophagy process provides an important new entry point toward developing therapies against age-related diseases. Recent studies in mammals have uncovered that the helix-loop-helix transcription factor EB (TFEB) is a central regulator of autophagy. We have reported that the C. elegans HLH-30 protein is a functional ortholog of TFEB and plays a broad role in C. elegans lifespan determination (Lapierre et al., Nature Communications, 2013). Specifically, we have found that HLH-30 is required for the lifespan extension observed in six independent longevity models, all of which are also known to be dependent on autophagy. Notably, HLH-30 is localized in the nucleus of intestinal cells in all of these long-lived mutants. Importantly, we have found TFEB to be similarly regulated in a mammalian longevity model suggesting that the longevity function of TFEB is also conserved. Therefore, we hypothesize that TFEB is a central player that modulates aging, at least in part by coordinating autophagic flux and, as such, represents an attractive longevity factor to molecularly understand in order to design approaches to promote healthspan and prevent aging. To address this hypothesis, we propose to use a combination of cutting-edge genetics and biochemical approaches to understand the spatial- and nuclear requirements for HLH-30 function in C. elegans lifespan (Aim 1); to address if downstream target genes besides those identified to function in autophagy are critical for longevity (Aim 2); and, finally, to identify genetic and pharmacological regulators of autophagy and HLH- 30/TFEB function (Aim 3). These studies are significant as they are the first to identify novel genetic regulators and post-translational modifications of HLH-30/TFEB in an unbiased fashion, and they will generate important information for future analysis of mammalian TFEB, a central regulator of autophagy as well as metabolism, in organismal aging. Furthermore, our studies are innovative because we employ C. elegans and mammalian cells in parallel to rapidly identify novel, conserved genes with roles in aging and to develop pharmacological solutions to enhance autophagy in order to prevent age-related diseases.